EGD1 (β-NAC) mRNA is localized in a novel cytoplasmic structure in Saccharomyces cerevisiae

RNA localization is a common mechanism for recruiting proteins to specific regions of a cell, which causes cell polarization and sometimes asymmetric division. We found that EGD1 mRNA accumulates dose-dependently as a cytoplasmic granule in Saccharomyces cerevisiae. EGD1 encodes a β-subunit of the nascent polypeptide-associated complex (NAC). NAC is a heterodimer consisting of α- and β-subunits, associated with ribosomes and thought to be involved in the folding of nascent polypeptide chains. Analysis of deletion constructs showed that the localization of EGD1 mRNA requires both an upstream region and an ORF of EGD1, suggesting that the translation of Egd1p is important for localization. We also showed that Egd1p and P-body components are co-localized with EGD1 mRNA. This granule, named the EGD1 granule, has features similar to cellular inclusions containing aggregated proteins. Disruption of microtubules by treatment with a drug, benomyl, resulted in loss of the EGD1 granule. When the expression level of EGD2 encoding the αNAC increased, the percentage of cells showing the EGD1 granule decreased, suggesting that the granular distribution of EGD1 depends on the quantitative balance between α- and β-subunits of NAC. Taken together, we propose a novel microtubule-dependent mechanism for controlling NAC through RNA localization.     

Messenger RNA 3′-end formation of a DNA fragment from the human c-myc 3′-end region in Saccharomyces cerevisiae

We have tested the functioning of the human c-myc polyadenylation signal in Saccharomyces cerevisiae. A DNA fragment containing the two AATAAA polyadenylation signals of the c-myc gene was inserted into a plasmid designed for the in-vivo testing of polyadenylation signals in yeast. The c-myc fragment had a partial capacity for directing mRNA 3-end formation in yeast. The 3-endpoints were 50–100 bp distant from the mRNA 3-ends mapped in humans. This human DNA fragment is therefore unspecifically functional in yeast, indicating that other sequence elements than the human polyadenylation signal, AATAAA, are necessary for 3-end formation.     

Blockage to exonuclease III digestion in the chromatin of Saccharomyces cerevisiae maps to the in vitro — determined binding site of a trans-acting regulatory factor

Summary A series of transposable element-induced mutations at the HIS4 locus in Saccharomyces cerevisiae have been attributed to the transposition of a Ty element into the 5 regulatory region of this gene. Various Ty-containing His⁺ revertants have been isolated and the HIS4/Ty junction region sequenced. The only difference found in this region between a His^- and a weak His⁺ strain was a single point mutation, an AG transition. The position of Ty remained unaltered. Examination of lacZ fusion plasmids further implicated this AG transition as being reponsible for the altered phenotype, the bp transition representing an allele of a cis-acting regulatory element. Subsequent gel retardation and methylation interference experiments revealed that this AG mutation enabled the binding of a trans-acting factor (TyBf) in vitro. In this paper we show that the TyBf binding site is in a region of chromatin hypersensitive to digestion by DNase I. The binding site is protected in vivo from digestion with exonuclease III, suggesting the presence of a bound protein in His⁺ (on) but not His^- (off) Ty-containing strains. We propose that a trans-acting factor binding in vivo, presumably TyBf, is responsible for the activation of HIS4 expression in these insertion mutants.     

POL32, a subunit of the Saccharomyces cerevisiae DNA polymerase δ, defines a link between DNA replication and the mutagenic bypass repair pathway

Pol32 is a subunit of Saccharomyces cerevisiae DNA polymerase δ required in DNA replication and repair. To gain insight into the function of Pol32 and to determine in which repair pathway POL32 may be involved, we extended the analysis of the pol32Δ mutant with respect to UV and methylation sensitivity, UV-induced mutagenesis; and we performed an epistasis analysis of UV sensitivity by combining the pol32Δ with mutations in several genes for postreplication repair (RAD6 group), nucleotide excision repair (RAD3 group) and recombinational repair (RAD52 group). These studies showed that pol32Δ is deficient in UV-induced mutagenesis and place POL32 in the error-prone RAD6/REV3 pathway. We also found that the increase in the CAN1 spontaneous forward mutation of different rad mutators relies entirely or partially on a functional POL32 gene. Moreover, in a two-hybrid screen, we observed that Pol32 interacts with Srs2, a DNA helicase required for DNA replication and mutagenesis. Simultaneous deletion of POL32 and SRS2 dramatically decreases cellular viability at 15 °C and greatly increases cellular sensitivity to hydroxyurea at the permissive temperature. Based on these findings, we propose that POL32 defines a link between the DNA polymerase and helicase activities, and plays a role in the mutagenic bypass repair pathway. Received: 25 May 2000 / Accepted: 3 July 2000     

The Use of 3, 3′, 5, 5′-Tetramethylbenzidine as a Peroxidase Substrate in Microplate Enzyme-Linked Immunosorbent Assay

Abstract

The assay conditions for the use of 3)3′,5,5′ tetramethylbenzidine (TMB) in microplate enzyme-linked immunosorbent assay are described. TMB is a safe (non-mutagenic) chromogen that is more sensitive than OPD as a substrate for horseradish peroxidase. We describe the optimum storage and assay conditions for this chromogen.     

Characterization of a novel α-amylase from Lipomyces kononenkoae and expression of its gene (LKA1) in Saccharomyces cerevisiae

A highly active -amylase (76 250 Da) secreted by the raw starch-degrading yeast Lipomyces kononenkoae strain IGC4052B was purified and characterized. Using high performance liquid chromatography (HPLC), end-product analysis indicated that the L. kononenkoae -amylase acted by endo-hydrolysis on glucose polymers containing -1,4 and -1,6 bonds, producing mainly maltose, maltotriose and maltotetraose. The following NH₂-terminal amino acids were determined for the purified enzyme: Asp-Cys-Thr-Thr-Val-Thr-Val-Leu-Ser-Ser-Pro-Glu-Ser-Val-Thr-Gly. The L. kononenkoae -amylase-encoding gene (LKA1), previously cloned as a cDNA fragment, was expressed in Saccharomyces cerevisiae under the control of the PGK1 promoter. The native signal sequence efficiently directed the secretion of the glycosylated protein in S. cerevisiae. De-glycosylation of the enzyme indicated that post-translational glycosylation is different in S. cerevisiae from that in L. kononenkoae. Zymogram analysis indicated that glycosylation of the protein in S. cerevisiae had a negative effect on enzyme activity. Southern-blot analysis revealed that there is only a single LKA1 gene present in the genome of L. kononenkoae.     

Healing for destruction: tRNA intron degradation in yeast is a two-step cytoplasmic process catalyzed by tRNA ligase Rlg1 and 5′-to-3′ exonuclease Xrn1

In eukaryotes and archaea, tRNA splicing generates free intron molecules. Although ∼600,000 introns are produced per generation in yeast, they are barely detectable in cells, indicating efficient turnover of introns. Through a genome-wide search for genes involved in tRNA biology in yeast, we uncovered the mechanism for intron turnover. This process requires healing of the 5′ termini of linear introns by the tRNA ligase Rlg1 and destruction by the cytoplasmic tRNA quality control 5′-to-3′ exonuclease Xrn1, which has specificity for RNAs with 5′ monophosphate.     

Pbp1 mediates the aberrant expression of genes involved in growth defect of ccr4∆ and pop2∆ mutants in yeast Saccharomyces cerevisiae

CCR4 and POP2 genes encode the catalytic subunit of the Ccr4-Not complex involved in shortening mRNA poly(A) tail in Saccharomyces cerevisiae. The ccr4Δ and pop2∆ mutants exhibit pleiotropic phenotypes such as slow and temperature-sensitive growth, aberrant expression of glucose repression genes and abnormal cell wall synthesis. We previously found that the growth defect of the ccr4Δ and pop2∆ mutants is suppressed by deletion of the PBP1 gene, which encodes poly(A)-binding protein (Pab1)-binding protein 1. In this study, we investigated the functional relationship between Ccr4/Pop2 and Pbp1 by measuring changes in gene expression in ccr4Δ and pop2∆ single mutants and ccr4Δ pbp1∆ and pop2∆ pbp1∆ double mutants. We found that expression of HSP12, HSP26, PIR3, FUS1 and GPH1 was increased in ccr4Δ and pop2∆ single mutants. The pbp1∆ mutation not only restored the growth defect but also reduced the increased expression of those genes found in the ccr4Δ and pop2∆ mutants. Over-expression of PBP1 in the ccr4Δ mutant further increased the expression of HSP12, HSP26, PIR3 and FUS1 and exacerbated the cell growth. These results suggest that the aberrant expression of a subset of genes, which is facilitated by Pbp1, contributes to the pleiotropic phenotypes of the ccr4Δ and pop2∆ mutants.     

Increased sensitivity of B-cell clonality analysis in formalin-fixed and paraffin-embedded B-cell lymphoma samples using an enzyme blend with both 5′→3′ DNA polymerase and 3′→5′ exonuclease activity

Polymerase chain reaction (PCR)-based detection of immunoglobulin heavy chain (IgH) gene rearrangement for determination of B-cell clonality needs to be simple but optimally sensitive. Efficient IgH PCR analysis can be hampered by sequence variability in the template DNA, despite of the use of degenerative primers. To improve sensitivity of the B-cell clonality analysis in formalin-fixed and paraffin-embedded (FFPE) tissues, we have performed framework three-area (FR3)/joining gene (JH) IgH PCR utilizing an enzyme blend (rTth DNA Polymerase, XL) providing both 53 polymerase and 35 exonuclease activities. The DNA samples were extracted from FFPE biopsies of 43 mature B-cell lymphoma cases of so-called germinal center and post-germinal center origin, including 6 nodal follicular lymphomas (FL), 15 gastric mucosa-associated lymphoid tissue (MALT) lymphomas, and 22 gastric diffuse large B-cell lymphomas (DLBCL). Of the cases, 31 (17 DLBCL and 14 MALT lymphoma) represented small endoscopic biopsies. Serial dilutions of target DNA were applied to avoid inconsistent bands that may be seen when the input amount of template is too low, which can be the case when DNA is extracted from FFPE endoscopic gastric biopsies. Using conventional Taq polymerase, consistent monoclonal product was found in 53% (23/43) of the cases (FL: 67%; MALT lymphoma: 47%; DLBCL: 55%). The rTth polymerase showed reproducible monoclonal pattern in 72% (31/43) of the cases (FL: 67%; MALT lymphoma: 73%; DLBCL: 73%); the sensitivity is compatible with one that can be detected with conventional FR3/JH PCR in fresh/frozen tissues. In conclusion, the rTth DNA polymerase greatly improves sensitivity of FR3/JH PCR in FFPE biopsies of mature B-cell lymphomas, most probably by increasing the primer matches during PCR amplification.     

Induction of an ATP-polymerizing enzyme in TMV-infected tobacco and its homology to the human 2′–5′ a synthetase

Several reports have indicated that tobacco carries an enzyme (APE) that, in the presence of poly (rI):(rC), polymerizes ATP to oligoadenylates. This paper demonstrates that the tobacco APE system comprises several proteins (estimated sizes: 32, 42, 67, and 84±10% kD). Only one of these proteins (the 67-kD form) binds to poly (rI):(rC). This APE form has been purified by affinity chromatography on a synthetic ds-RNA column. Four tobacco proteins, including the purified one, crossreact with antibodies against the human enzyme, 2–5 A synthetase. The ATP-binding capacity of some of these proteins has also been demonstrated. The amount of plant oligoadenylates obtained by polymerizing ATP with the purified APE form allows, for the first time, their direct analysis by TLC. The TLC analysis indicated that the oligomer produced by APE is not identical to the 2–5 oligoadenylate.     

Evaluation of sequencing of HCV core/E1, NS5A and NS5B as a genotype predictive tool in comparison with commercial assays targeting 5′UTR

BackgroundHepatitis C virus (HCV) genotyping is required for tailoring the dose and duration of antiviral therapy, predicting virological response rates, and selecting future treatment options.ObjectiveTo establish whether baseline genotypes, performed by INNO-LiPA Version 1.0 (v1.0), before 2008, were valid for making treatment decisions now or whether genotypic determination should be repeated. Furthermore, to evaluate concordance between Abbott RealTime genotype II assay (RT) and genotyping by sequencing HCV C/E1, NS5A, NS5B.Study designGenotyping by RT and sequencing was performed on paired historic and current specimens from 50 patients previously baseline genotyped using INNO-LiPA.ResultsOf 100 samples from 50 patients, ≥2 of HCV genomic target regions yielded a sequence that was suitable for genotyping, with 100% concordance, providing no evidence of recombination events. Genotype and subtype prediction based on RT and sequencing agreed in 62.8% historic and 72.7% current specimens, with a kappa coefficient score of 0.48 and 0.76, respectively.LiPA could not subtype 46% of HCV gt1 infections, and LiPA subgenotype was only in agreement with RT and sequencing in 28.6% cases, where matched baseline and historic specimens were available.Three patients were indeterminate by RT, and five patients with HCV gt1 infections could not be subtyped by RT. However, RT revealed mixed infections in five patients where sequencing detected only single HCV infection at 20% threshold.ConclusionGenotyping by sequencing, exhibited excellent concordance, with moderate to good agreement with RT, and could resolve RT indeterminates and subtype HCV-gt1 infections not possible by LiPA.     

Association of Variants in PPARγ2, IGF2BP2, and KCNQ1 with a Susceptibility to Gestational Diabetes Mellitus in a Korean Population

Purpose

Patients with gestational diabetes mellitus (GDM) have been reported to exhibit the same genetic susceptibility as that observed in those with type 2 diabetes mellitus (T2DM). Recent polymorphism studies have shown that several genes are related to T2DM and GDM. The aim of this study was to examine whether certain candidate genes, previously shown to be associated with T2DM, also offer a specific genetic predisposition to GDM.

Materials and Methods

The current study was conducted in 136 Korean pregnant women, who gave birth at Gil Hospital, from October 2008 to May 2011. These study subjects included 95 subjects with GDM and 41 non-diabetic controls. We selected the specific genes of PPARγ², IGF2BP2, and KCNQ1 for study and amplified them using the polymerase chain reaction. This was followed by genotyping for single nucleotide polymorphisms. We then compared the genotype frequencies between patients with GDM and non-diabetic controls using the χ² test. We obtained and analyzed clinical information using Student''s t-test, and statistical analyses were conducted using logistic regression with SPSS Statistics software, version 19.0.

Results

Significant differences were observed in maternal age, body mass index, weight gain and weight at time of delivery between the groups compared. Among pregnant women, polymorphisms in PPARγ² and IGF2BP2 were shown to be highly correlated with GDM occurrence, whereas no correlation was found for KCNQ1 polymorphisms.

Conclusion

Our results indicated that genetic polymorphisms could also be of value in predicting the occurrence and diagnosis of GDM.     

Sensitivity of brome mosaic virus RNA1 replication to mutations in the 3′ tRNA-like structure implies a requirement for sustained synthesis of replicase protein 1a

Summary. The replication competence of a series of brome mosaic virus (BMV) RNA1 variants with defined mutations in the 3′ tRNA-like structure, previously characterized in vitro to be defective in minus-strand synthesis and several tRNA-associated functions, was analyzed in barley protoplasts. Inocula containing wild type RNAs2 and 3 and RNA1 bearing either Δknob or 5′Psk mutation failed to replicate. Two additional RNA1 variants, each bearing either M4 or 5′AGA mutation, resulted in detectable accumulation of progeny but are inhibitory to overall viral replication when supplied in high concentrations. Another aminoacylation-defective mutation Δ5′ supported viral replication but did not interfere with viral replication even at higher concentrations. Coinoculation of replication-incompetent variants of RNAl with wt RNAs2 and 3 to Chenopodium hybridum plants resulted in the delayed development of local necrotic lesions characteristic of a wt infection. Sequence analysis of progeny RNA recovered from these lesions indicated that, in each case, a functional 3′ noncoding sequence was restored due to homologous recombination with a corresponding sequence from wt RNA3. Taken together the results suggest that, unlike protein 2a which is required in catalytic amounts, the intrinsic involvement of protein 1a at various stages of virus infection cycle demands its sustained synthesis.     

Genetic polymorphisms in MDR1, CYP3A4 and CYP3A5 genes in a Ghanaian population: a plausible explanation for altered metabolism of ivermectin in humans?

Background  

Ivermectin, a substrate of multidrug resistance (MDR1) gene and cytochrome P450 (CYP) 3A4, has been used successfully in the treatment of onchocerciasis in Ghana. However, there have been reports of suboptimal response in some patients after repeated treatment. Polymorphisms in host MDR1 and CYP3A genes may explain the observed suboptimal response to ivermectin. We genotyped relevant functional polymorphisms of MDR1 and CYP3A in a random sample of healthy Ghanaians and compared the data with that of ivermectin-treated patients with a view to exploring the relationship between suboptimal response to ivermectin and MDR1 and CYP3A allelic frequencies.